This invention relates to reagents for determining .beta.-1,3-glucan or peptidoglycan and a method for determining .beta.-1,3-glucan or peptidoglycan using the same.
.beta.-1,3-Glucan (hereinafter referred to as ".beta.-G") is present in nature as cell wall components of true fungi such as yeast and molds and as major polysaccharide components in fruit bodies of many basidiomycota. It is well known that .beta.-G is positive in the so-called Limulus test which is a method for detecting endotoxin using a reagent purified from horseshoe crab hemocyte lysate. By applying this property of .beta.-G, a method is proposed for determining .beta.-G specifically with no influence of endotoxin, in which a reagent purified from horseshoe crab hemocyte lysate is used [Rinsho Byori (Clinical Pathology) 33, 639-644, 1985]. However such a method has not practically been used yet, because of a trouble such as a stability of the reagent. Further, there is proposed a method for detecting endotoxins or .beta.-G by using a reagent obtained by purifying a blood cell lysate from a crustacean or an insect [PCT, International Publication No. WO 83/02123]. But the reagent used for this process has a property to react with both the endotoxins and .beta.-G. Therefore, in order to use this reagent for determining specifically .beta.-G, it is necessary to add anti-endotoxin factors purified from crayfish blood cells.
On the other hand, peptidoglycan (hereinafter referred to as "PG") is a kind of glycopeptide polymer constituting cell walls of bacteria and is contained more in cell walls of Gram-positive bacteria than in cell walls of Gram-negative bacteria. In general, PG is characterized by containing N-acetyl- or N-glycolylmuramic acid and D-amino acid. It has been known that PG has many biological activities such as having an attack of fever, lowering of functions of the liver and the kidney, enhancing the action of endotoxin activity, increasing effect of immune function (adjuvant activity) and the like. Thus, studies of PG have been made vividly in the fields of medical science, pharmacology, microbiology, etc., but a specific determination method has not been found.
Some of the present inventors previously found that a body fluid obtained from silkworm does not react with endotoxin but reacts with PG or .beta.-G so as to activate at least three kinds of enzymes, i.e. an esterase hydrolyzing N-.alpha.-benzoyl-L-arginine ethyl ester (BAEEase), prophenoloxidase activating enzyme (PPAE), and phenoloxidase (PO) [Insect Biochem, vol 16, No. 3, pp. 539-545, 1986]. But since there was a problem in specificity, this was not applied to the determination of .beta.-G or PG.
As mentioned above, since the determination methods are not established, effects of .beta.-G or PG on human bodies are not fully known yet. But recently, there is a doubt that .beta.-G is one cause for bringing about a shock at the time of using hemodicalysis film of cellulose derivatives, or there is a suggestion that .beta.-G is present in a body fluid of a patient suffered from mycotic infection. Therefore, the determination of .beta.-G or PG becomes more and more important in the fields of medical sciences, such as pharmacology and microbiology.